Production of staphylococcal enterotoxin in mixed cultures

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Production of staphylococcal enterotoxin in mixed cultures.

Two Staphylococcus aureus strains were grown in brain-heart infusion (BHI) broth and a meat medium with Bacillus cereus, Streptococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Both S. aureus strains grew well and produced enterotoxin in the presence of S. faecalis in BHI broth; however, enterotoxin production was observable in the meat medium only when the S. aureus inoculum was greater than the S. faecalis inoculum. S. aureus FRI-100 grown with B. cereus produced enterotoxin in both media only when the S. aureus inoculum was much higher than the B. cereus inoculum (10 versus 10(4) CFU), whereas S. aureus FRI-196E produced enterotoxin in both media at all inoculum combinations except in the meat medium, when the inocula of the two organisms were the same. S. aureus grown with E. coli in BHI broth produced enterotoxin at all inoculum combinations except when the E. coli inoculum was greater than the S. aureus inoculum; however, in the meat medium, enterotoxin was produced only when the S. aureus inoculum was much greater than the E. coli inoculum (10 versus 10(4) CFU), S. aureus FRI-100 grown with P. aeruginosa in either medium produced enterotoxin only when the S. aureus inoculum was much greater than the P. aeruginosa inoculum (10 versus 10(3) or 10(4) CFU). It can be concluded from these results that enterotoxin production is unlikely in mixed cultures unless the staphylococci outnumber the other contaminating organisms.     

Injury of Bacteria by Sanitizers

Injury of test cultures was quantitated by differences in colony counts obtained with a complete medium and those obtained on conventional selective media. Staphylococcus aureus, Streptococcus faecalis, and several strains of Escherichia coli were injured when exposed to the quaternary ammonium compound methylalkyltrimethyl ammonium chloride. Representative hypochlorite sanitizers also caused injury of E. coli ML30. Sanitizer concentration appeared to be the main factor in the cause of death and injury, a higher concentration being needed to cause death. Increases in temperature did not result in substantial increases in injury; however, the lethal effect was greater at higher temperatures. Varying the cell concentration from 10(7) to 10(9) cells per ml did not change the fraction of cell population killed or injured. The inability or failure of common selective media to detect injured bacteria in food could have serious public health consequences.     

Effect of Salt Concentration in the Recovery Medium on Heat-injured Streptococcus faecalis

Properties relating to the recovery of three heat-injured strains of Streptococcus faecalis were studied. All strains were cultured in all purpose plus Tween broth (APT) at 30 C for 24 hr before being subjected to heat in fresh APT broth. APT recovery medium containing various added amounts of NaCl, KCl, MgCl(2), or KCl and MgCl(2) was used to assess the effect of salts on the recovery of thermally injured S. faecalis. It was evident that, upon exposure to heat, S. faecalis cells became sensitive to increased salt concentrations. Analyses to determine the ribonucleic acid (RNA) content of heated cells showed a reduction of cellular RNA, but the per cent reduction was not directly proportional to the per cent reduction of the viable cells.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coliform bacteria on three occasions during 1983. Eighty percent or more of the coliform bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherichia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 micrograms/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coli-form bacteria on three occasions during 1983. Eighty percent or more of the coli-form bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherchia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 μg/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

Sensitivity of Legionella pneumophila to sunlight in fresh and marine waters.

Studies were carried out to assess the sunlight sensitivity of Legionella pneumophila suspended in fresh and marine waters. Comparison studies on sunlight sensitivity of lake water bacteria, Pseudomonas aeruginosa, Escherichia coli and Streptococcus faecalis, were also undertaken. The effects of full sunlight and polyacrylic-screened sunlight were monitored in the study. Results indicate that L. pneumophila cells are slightly more sensitive to sunlight in seawater than in fresh water. Enumeration of sunlight-stressed bacteria in fresh water was found to be dependent on the medium used, and the following order of sensitivity to sunlight, from least to most sensitive, was noted: natural lake water bacteria, L. pneumophila, P. aeruginosa, E. coli, and S. faecalis.     

Factors Influencing the Occurrence of High Numbers of Iodine-Resistant Bacteria in Iodinated Swimming Pools

It has been shown that, although iodinated swimming-pool waters are usually free from coliform bacteria and enterococci, the total counts frequently become relatively high. Pseudomonas alcaligenes and Alcaligenes faecalis have been shown to account for most of these high counts. It was of interest, therefore, to compare the microbial flora of four alternately chlorinated and iodinated swimming pools. By means of the membrane filter method and suitable selective media, examinations were made for total viable counts, coliform bacteria, enterococci, staphylococci, Streptococcus salivarius, and P. aeruginosa. Colonies also were picked from membrane filters incubated on standard plate count agar and identified. The results showed that, although viable counts were significantly higher during the iodinated periods, the specific types of bacteria determined were either fewer than or the same as in chlorinated periods. During chlorination, the predominant microbial flora consisted of staphylococci and members of the genus Bacillus. During iodination, however, the P. alcaligenes-A. faecalis group accounted for 92 to 99% of the microbial flora. The accumulation of high numbers of these bacteria was shown to be due to their iodine resistance and their ability to grow rapidly in pool water in the absence of free iodine.     

Cell adsorption and selective desorption for separation of microbial cells by using chitosan-immobilized silica

Cell adsorption and selective desorption for separation of microbial cells were conducted by using chitosan-immobilized silica (CIS). When chitosan was immobilized onto silica surfaces with glutaraldehyde, bacterial cells adsorbed well and retained viability. Testing of the adsorption and desorption ability of CIS using various microbes such as Escherichia coli, Aeromonas hydrophila, Pseudomonas aeruginosa, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus casei, Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Saccharomyces cerevisiae, Saccharomyces ludwigii, and Schizosaccharomyces pombe revealed that most microbes could be adsorbed and selectively desorbed under different conditions. In particular, recovery was improved when L-cysteine was added. A mixture of two bacterial strains adsorbed onto CIS could also be successfully separated by use of specific solutions for each strain. Most of the desorbed cells were alive. Thus, quantitative and selective fractionation of cells is readily achievable by employing chitosan, a known antibacterial material.     

Transformation and fusion of Streptococcus faecalis protoplasts.

Nonconjugative plasmids were transferred by protoplast fusion among Streptococcus faecalis strains and from Streptococcus sanguis to S. faecalis. S. faecalis protoplasts were also transformed with several different plasmids, including the Tn917 delivery vehicle pTV1. Transformation was reproducible, but low in frequency (10(-6) transformants per viable protoplast). A new shuttle vector (pAM610), able to replicate in Escherichia coli and S. faecalis, was constructed and transformed into S. faecalis protoplasts. pAM610 was mobilized by the conjugative plasmid pAM beta 1 in matings among S. faecalis strains and from S. sanguis to S. faecalis. Chimeric derivatives of pAM610 were also transformed into S. faecalis.     

Thermal Injury and Recovery of Streptococcus faecalis

Exposure of Streptococcus faecalis R57 to sublethal heating produced a temporary change in the salt tolerance and growth of the organism. After sublethal heat treatment at 60 C for 15 min, greater than 99.0% of the viable population was unable to reproduce on media containing 6% NaCl. In addition, the heated cells displayed a sensitivity to incubation temperature, pH, and 0.01% methylene blue. When the injured cells were placed in a synthetic medium, recovery occurred at a much slower rate than in a complex medium. However, both media supported comparable growth of the uninjured organism. Various media used for the enrichment of streptococci also provided a suitable environment for the recovery of the injured cells. Generally, as more selective agents were present in the medium, the rates of recovery decreased. Metabolic inhibitor studies with chloramphenicol, penicillin, and actinomycin D substantiated the fact that the process involved was recovery and not growth, and that this recovery was linked to ribonucleic acid synthesis.     

Effect of Storage at 1° and 4°C on Viability and Injury of Staphylococcus aureus, Escherichia coli and Streptococcus faecalis

Staphylococcus aureus and Escherichia coli incubated at 1° or 4°C became increasingly sensitive to Mannitol Salt Agar and Violet Red Bile Agar respectively. The increased sensitivity was most marked with exponential phase cultures. Streptococcus faecalis was refractory to similar treatments. The viability of all 3 organisms on selective and non-selective media is reported.     

Water-in-oil macroemulsions sustain long-term viability of microbial cells in organic solvents

Extremely stable water-in-oil macroemulsions have been obtained by dispersing water in isooctane in the presence of lecithin. Either prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and Rhodotorula minuta) cells hosted in these water-in-oil macroemulsions are viable for weeks despite the consistent excess of organic solvent (ranging from 70 to 84%, v/v) in these ternary systems. Conjugation occurs upon mixing macroemulsions containing F(+) or F(-) Escherichia coli strains, indicating consistent mass transfer between the water droplets. Populations of yeasts hosted in water-in-oil macroemulsion feature a higher frequency of cells aggregation when compared with the corresponding populations suspended in homogeneous aqueous media.     

Growth of starved Escherichia coli O157 cells in selective and non-selective media.

Escherichia coli O157 strains starved in sterile deionized water (SDW) and filter-sterilized natural river water (SRW) were investigated with specific reference to their culturability in selective and non-selective media. Growth of the strains starved in both SDW and SRW were markedly suppressed with time in selective liquid media such as modified trypticase soy broth supplemented with novobiocin (mTSB+n) and modified E. coli broth supplemented with novobiocin (mEC+n). This suppression was more pronounced when incubated at 42 C than at 37 C, especially with mEC+n. By contrast, such growth suppression was seldom observed when cultured at 37 C in non-selective liquid media such as trypticase soy broth (TSB) and buffered peptone water. In mEC+n at 42 C, the non-starved cells from overnight cultures with an initial density of less than 10(3) colony-forming units (CFU)/ml grew to the density of over 10(7) CFU/ml after 24 hr incubation, whereas those starved for 6 weeks in SRW were only to maintain their initial density or died off after 24 hr incubation under the same culturing conditions. These results indicated that the isolation of starved cells of E. coli O157 from water samples would be most difficult with selective enrichment or direct plating on the selective plate media. It is thus highly recommended that a "resuscitation" of the cells with non-selective enrichment should be performed as a routine practice for maximum recovery of E. coli O157 from water systems.     

Removal of bacteria from water by adhesion to cross-linked poly(vinylpyridinium halide).

Cross-linked poly(vinylpyridinium halide) was found to have a novel and remarkable ability to remove bacteria from water. For example, when 10 g (wet weight) of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) was contacted with 20 ml of suspensions of Escherichia coli (9.7 X 10(4) to 9.7 X 10(7)/ml), Salmonella typhimurium (8.0 X 10(6) to 1.1 X 10(7)/ml), Streptococcus faecalis (5.0 X 10(7)/ml), Staphylococcus aureus (8.1 X 10(7)/ml), and Pseudomonas aeruginosa (3.2 X 10(5)/ml) under stirring in sterilized physiological saline at 37 degrees C, 99% of the viable cells of these bacteria were removed in 2 to 6 h. When suspensions of these bacteria (10(5) to 10(8) cells per ml) were passed through a column (20 mm by 100 cm) of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) at 37 degrees C with a flow rate of 0.8 to 1.4 bed volumes per h, 97 to 100% of the viable cells were eliminated from the suspensions during the treatment. Mechanistic studies demonstrated that cross-linked poly(vinylpyridinium halide) irreversibly captured these bacteria alive during the treatment. That is, total organic carbon was removed during the treatment, and the bacteria which adhered to the resin proliferated on the bacterial medium. The adhesion capacity was estimated to be 10(10) cells per g (dry weight). Total organic carbon was also removed even when the bacteria were killed by heat treatment before the column studies.     

Dependence of Protease Secretion by Streptococcus faecalis var. liquefaciens on Arginine and Its Possible Relation to Site of Synthesis

Washed cells of Streptococcus faecalis var. liquefaciens, when harvested from media that supported protease biosynthesis, continued to release this extracellular enzyme in phosphate buffer. The addition of ethylenediaminetetraacetic acid (EDTA) halted the secretion. If zinc ions were added to the EDTA-treated cells before 45 to 60 min had elapsed, a fraction of the anticipated enzyme activity was observed. After 60 min, arginine and phosphate, in addition to zinc, were necessary for the demonstration of proteolytic activity. The enzyme that was released was newly formed, because chloramphenicol, puromycin, or actinomycin D prevented its appearance. Energy for this synthetic reaction was obtained, apparently, from arginine; lactose could not be substituted for arginine. This last point is interpreted to mean that extracellular protease biosynthesis occurs in a localized area or cellular compartment into which the adenosine triphosphate derived from the fermentation of lactose cannot diffuse. No evidence was found for a protease zymogen, although this possibility is not completely precluded.     

Biosurfactant production by thermophilic dairy streptococci

Biosurfactant production of eight Streptococcus thermophilus strains, isolated from heat exchanger plates in the downstream side of the regenerator section of pasteurizers in the dairy industry has been measured using axisymmetric drop shape analysis by profile (ADSA-P). Strains were grown in M17 broth with either lactose, saccharose or glucose added. After harvesting, cells were suspended in water or in 10 mm potassium phosphate buffer, pH 7.0, and suspension droplets were put on a piece of FEP-Teflon. Changes in droplet profile were analysed by ADSA-P to yield the surface tension decrease due to biosurfactant production as a function of time. Surface tension decreases larger than 8 mJ·m^–2 were taken as indicative of biosurfactant production. Only five strains produced biosurfactants in water, solely when saccharose was added to the growth medium. In buffer, all strains produced biosurfactants and production was generally greater than in water. Also, most strains suspended in buffer produced maximally when saccharose was added to the growth medium, whereas one strain produced maximally in buffer upon the addition of glucose. Four strains suspended in buffer produced biosurfactants when glucose was added and only two strains when lactose was added. The possible role of these biosurfactants as anti-adhesives in the dairy industry and for the survival of these strains in natural systems is discussed.Correspondence to: H. J. Busscher     

Species-identification and antimicrobial susceptibility tests by the fully automated RAISUS using an early-harvested cell suspension]

We evaluated the usefulness of an early-harvested bacterial cell suspension to the fully automated RAISUS (Nissui Pharmaceuticals Co., Ltd., Tokyo) to provide the results of species-identification and antimicrobial susceptibility testings within a day after overnight-incubation of the primary cultures. A single, well-separated colony appeared on the primary culture plate was transferred onto a blood agar or chocolate agar plates, then incubated for 3 to 6 hours. The cell suspension to the RAISUS was properly prepared to the McFarland 0.5 turbidity from the early-harvested bacterial cells. When the five ATCC reference strains, consisting of Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, were repeatedly tested for the species-identification, all the identification results were acceptable. Antimicrobial susceptibility tests were evaluated with the above five strains and Haemophilus influenzae ATCC 49247. The results obtained indicated that the most susceptibility test results were comparable to those MICs obtained by the standard test procedure, but some strains, in particular, H. influenzae and P. aeruginosa gave significantly discrepant MICs for certain antimicrobial agents. The significant discrepancy in MIC determinations regarded the difference of viable cell concentrations in the cell suspension prepared respectively. Through the analysis of laboratory workflow, it became to apparent that 18S to 20S of the tests were completed by 5:00 p.m., and it required to wait until 3:00 a.m. to complete 90S of the tests. With these results, the early-harvested bacterial cell suspension is applicable to species-identification by RAISUS, but it is necessary to adjust viable cell concentrations to antimicrobial susceptibility test. Also, it is urgent to reconstitute a daily workflow to improve the rapidity of RAISUS test function.     

Survival and detection of bacteria in an aquatic environment

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival and detection of bacteria in an aquatic environment.

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)